Fas is a 45kD transmembrane protein that belongs to the TNF receptor family. On activation, either by its ligand (FasL) or by antibody cross linking, Fas delivers a death signal resulting in apoptosis. In mice spontaneous loss of function mutations have been described in Fas (lpr) and FasL (gld). In both these mutant strains of mice, an abnormal accumulation of lymphocytes occurs suggesting that Fas and FasL are involved in normal lymphocyte death. This is consistent with recent observations that the Fas system is involved in the elimination of activated T cells after they have responded to foreign antigens. In the absence of a functioning Fas- FasL system, such as in lpr or gld mice, activated lymphocytes accumulate and contribute to the development of auto-immune disease. In the right genetic background (MRL) the lpr and gld mice develop a fatal lupus-like syndrome. Despite the importance of Fas in programmed cell death and auto-immunity, the molecular mechanism by which Fas kills remains an enigma. Fas, like other members of the TNF receptor family appears to possess no intrinsic signaling capacity (e.g., kinase activity), suggesting that signal transduction is likely mediated by associating molecules. Mutational analysis of the Fas receptor cytoplasmic domain has delineated a stretch of about 70 amino acids that is necessary and sufficient for transduction of the death signal (referred to as a death domain). To identify associated signal transducing molecules, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the Fas cytoplasmic domain. One such interacting protein, designated FADD (for Fas associated death domain), bound native Fas but not functionally inactive mutants. FADD itself contains a death domain homologous to the death domain of Fas suggesting a death domain to death domain interaction. Expression of FADD induces apoptosis. These findings suggest that FADD potentially plays an important role in the proximal signal transduction of Fas. Given this, the Specific Aims are as follows: Specific Aim 1: Confirm that endogenous FADD binds Fas and that the death domain is indeed responsible for receptor association. Additionally, use the yeast two-hybrid and complementary biochemical approaches to characterize FADD interacting proteins. Specific Aim 2: Using a dominant negative approach, determine if FADD is an intrinsic and necessary component of the Fas death pathway.